Biomarkers for inflammatory response

ABSTRACT

An in-vitro method for the prediction, prognosis and/or diagnosis of an inflammatory response associated with a condition or disease such as schizophrenia in a subject, the method comprising determining in a sample of a subject the level of 25-hydroxy vitamin D3, preferably in combination with the level of least one biomarker wherein the at least one biomarker is selected from innate chemokine (IL-8) and matrix metalloproteinase (MMP-9); and comparing the levels of said 25-hydroxy vitamin D3 and at least one biomarker to a control level of 25-hydroxy vitamin D3 and the at least one biomarker respectively in order to determine a positive or negative prediction, prognosis and/or diagnosis of said inflammatory response indicating an associated condition or disease, such as schizophrenia.

FIELD OF THE INVENTION

The present invention relates to a combinatorial biomarker assay forassessing inflammatory/immunological processes, particularly but notexclusively in schizophrenia and/or multiple sclerosis.

BACKGROUND OF THE INVENTION

Schizophrenia is a chronic severe psychiatric disorder that afflictsover 50 million people worldwide. Its major clinical features includethought disorders, auditory and visual hallucinations, delusions, socialwithdrawal, lack of motivation, and cognitive dysfunction. Its aetiologyis unknown, but it is now proving to be multi-factorial, with diversegenetic and environmental contributions. Identification of risk factorsshould hold important clues to novel preventive and therapeuticstrategies. Several aetiological theories have been proposed involvingdevelopmental or neurodegenerative processes, neurotransmitterabnormalities, infections, immune dysfunction and/or autoimmunemechanisms (1).

Evidence for contributions from the immune system comes from genome-wideassociation studies finding association with the majorhistocompatibility complex (MHC) region to schizophrenia (2).

Infection is one of the suspected risk factors for schizophrenia. Thefirst response to infection is mounted by the innate immune system andinflammation is a key part. Innate immune responses comprise severalreactions that isolate and destroy the invading pathogen.Pathogen-associated molecular patterns (PAMPs) are recognized by patternrecognition receptors (PRRs), which are expressed on antigen-presentingcells as proposed by Janeway in 1989 (12). The receptors includeToll-like receptors (TLRs), retinoic acid inducible gene (RIG-I)-likeand nucleoside oligomerisation domain (NOD)-like receptors. TLR-3, -7and -9 recognise microbial patterns inside infected cells, whereasTLR-2, -4 and -5 recognise bacterial patterns on cell surfaces. Oncestimulated, these receptors activate signalling pathways, which initiatethe secretion of innate chemokines/cytokines and help mount successfultailor-made adaptive anti-pathogen responses.

Early childhood infections of the brain increase schizophrenia risk˜5-fold (4, 5). Even during pregnancy, particularly the secondtrimester, infections correlate with an increased risk to offspringlater in life (5, 6). Several infections have been studied duringpregnancy such as rubella, influenza and toxoplasma (7). Injectingpregnant mice with synthetic double-stranded DNA poly I:C, to mimicviral infections/interferon responses, and lipopolysaccharide (LPS), ahighly inflammatory component of bacterial cell walls, elicitedmorphological and behavioral changes characteristic of the brain inschizophrenia (8-11); however, the underlying mechanisms of this changeare not fully understood.

Interestingly, fetal exposure to the innate chemokine IL-8, which ispart of the innate acute phase response, correlated significantly withschizophrenia risk in offspring (13). In addition, IL-8 levels inmothers during the second trimester of pregnancy correlatedsignificantly with increased schizophrenia risk in a prospective birthcohort study (14). Fetal exposure to elevated IL-8 triggered structuralneuroanatomic alterations, e.g. significant increases in ventricularcerebrospinal fluid, and significantly decreased volumes of the leftentorhinal and the right posterior cingulate cortex (15). Elevated IL-8levels have also been described in untreated schizophrenic patients exvivo and upon stimulation with LPS (16).

IL-8 is known to induce the expression of matrix metalloproteinase-9(MMP-9) in vitro (17), changes in which are implicated in schizophrenia,Alzheimer's disease, vascular dementia and also in multiple sclerosis,cancer and heart disease. A recent multi-analyte analysis ofcase-control collections found elevated MMP-9 levels in schizophrenia,which were not altered by neuroleptic treatment and may thereforerepresent a “trait” marker (18). Furthermore, recent genotyping studiesshowed associations with a functional polymorphism (-1562C/T) of theMMP-9 gene in schizophrenia (19), which was confirmed in a Chinesecohort (20), though not in a family-based association study (21).Interestingly, the same MMP-9 polymorphism may also influencesusceptibility to MS, where serum MMP-9 levels correlate withMRI-documented disease activity in relapsing-remitting disease (RRMS).The extracellular proteolytic system comprises metallo-proteinases andtheir endogenous tissue inhibitors (TIMPs). Some MMPs can processseveral of the proteins involved in synaptogenesis, synaptic plasticity,and long-term potentiation (22). MMP-9 regulates synaptic plasticity inthe hippocampus in vitro (23,24), and the ratio of TIMP-1 and MMP-9reportedly modulates learning and memory processes (25). Altered plasmaMMP-9 levels have also been described in Alzheimer's disease andvascular dementia (26). Interestingly, the same MMP-9 polymorphism alsoinfluenced susceptibility to MS (27), where serum MMP-9 levelscorrelated with MRI-documented disease activity in relapsing-remittingdisease (RRMS) (28).

MMP-9 may also play a role in defense against pathogens, wherestimulation with poly I:C leads to significant up-regulation of MMP-9levels in epithelial cells (29). In addition, MMP-9, and TIMP-1 weresignificantly higher in patients with HHV-6 encephalitis (30). MMP-9levels are also correlated with progressive liver damage in HBV and HCVinfection (31). Furthermore, MMP-9 is critical for effective bacterialphagocytosis of Streptococcus pneumoniae and reactive oxygen speciesgeneration in neutrophils (32). Stimulation of Toll-like receptor-4 withbacterial LPS increases MMP-9 expression in fibroblasts (33).

Of particular interest is the finding that MMP-9 gene expression,secretion and activity is significantly inhibited by vitamin-D in Mtuberculosis infection (34). Vitamin-D is produced by skin exposure toultraviolet B (UVB) solar radiation. UVB converts 7-dehydrocholesterolprecursors into previtamin D3, which spontaneously changes to vitamin-D3(VitD3). VitD3 is converted into 25-hydroxyvitamin D3 (25-OHD) and itsfinal active form, 1,25-dihydroxyvitamin D3 (1,25-OHD). Interestingly,there is an excess of schizophrenia patients born from January to March(35, 36), which might reflect maternal hypovitaminosis-D (37, 38),and/or maternal infection during pregnancy. A further study alsoidentified a >10 fold variation in schizophrenia prevalence and atendency for prevalence to increase with latitude (39), just as for MS,where VitD3 is also implicated (40). A recent case-control study onneonatal blood samples identified a significant association betweenneonatal vitamin-D status and risk of schizophrenia (41). In adultwomen, high intake of vitamin-D and fish correlated with a lower rate ofpsychotic-like symptoms (42). However, no significant association wasdetected between four vitamin-D receptor single nucleotide polymorphismsand the risk of schizophrenia (43). Nevertheless, low serum levels of25-OHD were reported among a psychiatric out-patient cohort in Sweden.In the Swedish schizophrenia cohort, the median 25-OHD was 45nmol/l—considerably lower than reported for healthy Swedish subjects.Only 14.5% had recommended levels of 25-OHD (>75 nmol/l).Hypovitaminosis-D was present in 56.5% of schizophrenia patients(levels<50 nmol/l) (44).

Currently, schizophrenia diagnosis is based on a clinician's ability tomake inferences about patients' inner experiences as no laboratory testsare available that aid in diagnosis, inform treatment strategies or helpmonitor and predict treatment response. Detrimental comorbiditiesinclude metabolic syndrome, other cardiovascular risk factors anddiabetes mellitus. Efficient biomarker discovery and assaying techniquesare therefore paramount, and will facilitate the future development ofpatient stratification and personalized medicine strategies.

Current treatments target neurotransmitter pathways in the brain.Antipsychotics act predominantly as dopamine D2 receptor antagonists,and target the symptoms but not the underlying cause of the disease.However, the exact mechanism leading to dopaminergic dysfunction inschizophrenia remains unknown. Recent findings highlight the role ofinflammation as an important player in dysregulation of theneurotransmitter system. As bacterial and viral infections are triggersof inflammatory responses, they are potential culprits in at least asubgroup of schizophrenia patients.

SUMMARY OF THE INVENTION

The inventors have identified a set of analytes as candidate blood-basedbiomarker signatures for schizophrenia, which mirror the existence ofdysregulated immune/inflammatory responses. They have surprisingly foundinterdependence between the inflammatory response in schizophrenia and25-hydroxyvitamin D and that low serum levels of 25-hydroxyvitamin-D3can be used as a biomarker of inflammation. Furthermore, they claim thatmeasuring several putative inflammatory biomarkers simultaneously as acombinatorial biomarker of active inflammation can significantly enhancediagnostic accuracy of the test in comparison to the measurement of asingle biomarker and offer intervention strategies. The analyte markersmay comprise additional cytokines/chemokines and Vitamin-D metabolites.

This invention highlights a novel and unique test to monitor peripheralinflammatory status using a combinatorial biomarker.

The present invention will allow monitoring of peripheral inflammation,and means of modulation in schizophrenia, other psychiatric/neurologicaland non-psychiatric/neurological conditions, e.g. mood disorders,Tourette syndrome, autism, Alzheimer's disease, amyotrophic lateralsclerosis, dementia, Parkinson's disease, multiple sclerosis in additionto persistent bacterial and viral infections, neuroAIDS, diabetes,metabolic syndrome, asthma, psoriasis, ulcerative colitis, rheumatoidarthritis and other autoimmune conditions, heart disease and cancer.

In the first aspect the invention provides an in vitro method for theprediction, prognosis or diagnosis of an inflammatory responseassociated with a particular disease or condition in a subject, themethod comprising determining in a biological sample of a subject thelevel of vitamin D3, preferably 25-hydroxyvitamin D3; and comparing thelevels of said vitamin D3, preferably 25-hydroxyvitamin D3 to a controllevel of vitamin D3, preferably 25-hydroxyvitamin D3 in order todetermine a positive or negative prediction, prognosis or diagnosis ofsaid inflammatory response associated with a disease or condition.

Preferably, the in vitro method is used to monitor peripheralinflammation. More preferably, the method provides for the prediction,prognosis or diagnosis of a condition or disease associated with aperipheral inflammatory response, such as those selected from the groupconsisting of schizophrenia, mood disorders, Tourette syndrome, autism,Alzheimer′ disease, amyotrophic lateral sclerosis, dementia, Parkinson'sdisease, multiple sclerosis and other psychiatric/neurological andnon-psychiatric/neurological conditions, neuroAIDS, diabetes, metabolicsyndrome, asthma, psoriasis, ulcerative colitis, rheumatoid arthritisand other autoimmune conditions, heart disease and cancer, preferablybeing schizophrenia and multiple sclerosis, especially schizophrenia.

More preferably, the method comprises determining in the biologicalsample the level of at least one biomarker in addition to the level ofvitamin D3, preferably 25-hydroxyvitamin D3, and comparing the level ofthe at least one biomarker to a control level of the biomarker in orderto determine a positive or negative prediction, prognosis or diagnosisof said inflammatory response. In particular, the at least one biomarkeris a cytokine or chemokine, more preferably the biomarker is selectedfrom innate chemokine (IL-8) and matrix metalloproteinase (MMP-9).

A specific embodiment of the invention provides a method for monitoringdisease activity/occurrence of comorbidities in schizophrenia comprisingmeasuring vitamin D3, especially 25-hydroxyvitamin D3 level in abiological sample from a subject in combination with the levels ofeither or both IL-8 and MMP-9 in the biological sample.

Preferably, the level of 25-hydroxyvitamin D3 indicative of a positivediagnosis is equal to or less than 75 nmol/L, more preferably equal toor less than 50 nmol/L. The level of IL-8 is preferably at least 20pg/ml, more preferably above 32 pg/ml compared to a control level belowthese values. The level of MMP-9 is preferably above 700 ng/ml, morepreferably above 705 ng/ml compared to a control level below thesevalues.

The invention also provides a method for determining the efficacy of atreatment regimen for treating an inflammatory response, in particularschizophrenia and/or multiple sclerosis, in a subject by comparing thelevels of 25-hydroxyvitamin D3 in a biological sample from the subject,preferably in combination with the levels of least one biomarker whereinthe at least one biomarker is selected from IL-8 and MMP-9 in thebiological sample before, during and after treatment, wherein saidtreatment is considered efficient if the level of 25-hydroxyvitamin D3and/or IL-8 or MMP-9 is approaching a predetermined control level forthe 25-hydroxyvitamin and/or IL-8 and/or MMP-9.

It is to be appreciated that determination of an efficient treatment isrecognised by measuring a level of 25-hydroxyvitamin D3 that is above 50nmol/L, preferably above 75 nmol/L relative to an initial measurementprior to treatment outside this range. The level of IL-8 is preferablybelow 20 pg/ml and the level of MMP-9 is preferably 700 ng/ml,preferably in the range 169-705 ng/ml, relative to an initialmeasurement prior to treatment that is outside this range

Another specific embodiment of the invention also provides a method forassessing the efficacy of a treatment regimen for treatingschizophrenia/monitoring of the occurrence of comorbidities in a subjectby comparing the levels of 25-hydroxyvitamin D3 in a biological samplefrom the subject in combination with the levels of IL-8 and MMP-9 in thebiological sample before, during and after treatment.

The present invention also provides a method for establishing areference biomarker profile comprising the steps of (a) determining aquantity of vitamin D3, preferably 25-hydroxyvitamin D3 and at least onebiomarker selected from IL-8 and MMP-9 in a sample obtained from ahealthy subject or a subject having an inflammatory response, especiallyhaving schizophrenia or multiple sclerosis; and (b) storing the quantityof the 25-hydroxyvitamin D3 and selected biomarker in a referencebiomarker profile respectively for healthy subjects or subjects havingan inflammatory response, especially having schizophrenia or multiplesclerosis.

The invention also relates to a kit comprising means for detecting thelevel of 25-hydroxyvitamin D3 in a biological sample from a subject,preferably in combination with the level of at least one biomarkerwherein the at least one biomarker is selected from IL-8 and MMP-9. Thekit comprises appropriate detection reagents and optionally furtheradditives.

An embodiment of the invention provides a kit comprising means fordetecting the level of 25-hydroxyvitamin D3 in a biological sample froma subject in combination with the levels of IL-8 and MMP-9 in thebiological sample.

In particular embodiments, the biological sample comprises serum,plasma, whole blood, saliva or urine. More preferably, the samplecomprises serum.

The subject is preferably a human subject and may or may not bediagnosed with a condition associated with a peripheral inflammatoryresponse, in particular schizophrenia.

The invention provides a rationale for down-regulating the inflammatoryresponse by vitamin-D3 supplementation to counteract hypovitaminosis-D.Inflammation is an important part of the innate immune system and in asubgroup of schizophrenia patients the activation of innate immunity bypathogens such as viruses and bacteria has to be considered and can betargeted by antibiotic or antiviral treatment.

Caren Ramien contributed to the experimental design and execution, dataanalysis and interpretation. Dr Sreeram Ramagopalan contributed to thedata interpretation and analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in further detail with reference tothe following examples and accompanying figures in which:

FIG. 1: Serum IL-8 levels in 16 schizophrenia patients (SCZ) and 10healthy controls (HC), determined by ELISA. The difference between themwas significant (29.17+/−17.66 pg/ml versus 1.88+/−0.52 pg/ml,p=0.0001). Data are expressed as means+/−SEM; p-values are derived froma two-tailed Mann-Whitney test.

FIG. 2: Serum levels of MMP-9 in SCZ patients and healthy controls(1200+/−120 ng/ml versus 130+/−18 ng/ml, p=0.0001), determined by ELISA.Data are expressed as means+/−SEM; p-values are derived from two-tailedMann-Whitney test.

FIG. 3: Correlating MMP-9 and 25-hydroxyvitamin D3 levels in 16 SCZpatients. The regression line is shown. Analysis by Spearman correlationyielded an r-value of −0.58 with p=0.017.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Investigation ObservingSignificantly Higher Levels of IL-8 in Schizophrenia Patients than inHealthy Controls

To investigate the potential role of the innate immune system in thepathophysiology of schizophrenia, we tested for an altered innateimmune/inflammatory signature(s) in patients' blood. As we had foundelevated levels of IL-8 in the serum of patients with multiple sclerosis(MS), and in experimental autoimmune encephalomyelitis (EAE) (45) (theanimal model of MS), we were interested in the IL-8 status ofschizophrenia patients (SCZ). We measured serum levels of IL-8 in SCZpatients and healthy controls (FIG. 1). They were significantly higherin SCZ patients than in healthy controls (p<0.05).

Example 2 Investigations Observed Significantly Elevated MMP-9 in SCZPatients than in Healthy Controls

We initially compared levels of MMP-9 in the serum of SCZ patients andhealthy controls. As shown in FIG. 2, the level of MMP-9 in SCZ patientswas significantly higher than in healthy controls (FIG. 2, p<0.05).

Example 3 Investigation into Hypovitaminosis-D in SCZ Patients

We next assessed the 25-hydroxyvitamin-D3 status of SCZ patients. Itsrecommended level is above 75 nmol/L (44) and levels under 50 nmol/Lmark deficiency (46). All but one patient were deficient in25-hydroxyvitamin-D3 (93.75%).

Example 4 Investigations Observed Significant Inverse Correlation of25-hydroxyvitaminD3 and MMP-9 Levels in SCZ Patients

In healthy controls, 25-hydroxyvitamin-D correlated inversely withcirculating MMP-9 (47). In our SCZ patients, we too found a significantinverse correlation (FIG. 3)—though not with IL-8 levels (data notshown).

Screening Assays: Measurement of MMP-9 and IL-8 Levels:

IL-8/CXCL8 and MMP-9 ELISAs (Qantikine Immunoassays R&D Systems, UK)were performed according to the manufacturer's instructions. SCZ andhealthy control samples were run on the same plate to control forinter-assay variation. All assays were performed in duplicate andsamples run in two separate assays. Absorbance reading was carried outon a Reader BioTek Synergy HT plate reader.

Measurement of 25-Hydroxyvitamin D3 Levels:

Serum levels of 25-hydroxyvitaminD3 were measured by isotope-dilutionliquid chromatography-tandem mass spectrometry as described previously(48).

Statistical Analysis

We used GraphPad Prism (5.00 version for Windows, GraphPad Software, SanDiego Calif. USA) for all statistical analyses. All analyses conductedused non-parametric tests, including Mann-Whitney tests to evaluatedifferences between groups, and Spearman rank correlations. P-valuesless than or equal to 0.05 were considered as statistically significant.

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1. An in vitro method for the prediction, prognosis and/or diagnosis ofan inflammatory response associated with a condition or disease in asubject, the method comprising determining in a biological sample of asubject the level of vitamin D3; and comparing the levels of saidvitamin D3 to a control level of vitamin D3 in order to determine apositive or negative prediction, prognosis and/or diagnosis of saidinflammatory response associated with a disease or condition.
 2. Amethod according to claim 1 wherein the level of 25-hydroxyvitamin D3 ismeasured and compared to a control level of this metabolite.
 3. A methodaccording to claim 1 for the prediction, prognosis and/or diagnosis of acondition or disease associated with the inflammatory response, saidcondition or disease being selected from the group consisting ofschizophrenia, mood disorders, Tourette syndrome, autism, Alzheimer′disease, amyotrophic lateral sclerosis, dementia, Parkinson's disease,multiple sclerosis in addition to persistent bacterial and viralinfections, neuroAIDS, diabetes, rheumatoid arthritis, metabolicsyndrome, asthma, ulcerative colitis, psoriasis and other autoimmuneconditions, heart disease and cancer and other psychiatric/neurological,non-psychiatric/neurological conditions
 4. A method as claimed in claim3, wherein the disease or condition is schizophrenia or multiplesclerosis.
 5. A method according to any one of claim 1 furthercomprising determining in the biological sample the level of at leastone biomarker in addition to the level of vitamin D3 and comparing thelevel of the at least one biomarker to a control level of the biomarkerin order to determine a positive or negative prediction, prognosis ordiagnosis of said inflammatory response.
 6. A method according to claim5 wherein the biomarker is selected from innate chemokine (IL-8) andmatrix metalloproteinase (MMP-9).
 7. A method according to claim 1wherein the level of vitamin D3, preferably 25-hydroxyvitamin D3,indicative of a positive inflammatory status is equal to or less than 75nmol/L, preferably under 50 nmol/L.
 8. A method according to claim 6wherein the level of IL-8 indicative of a positive inflammatory statusis at least 20 pg/ml, preferably at least 32 pg/ml and/or the level ofMMP-9 indicative of a positive inflammatory status is at least 700ng/ml, preferably at least 705 ng/ml.
 9. A method according to claim 1further comprising monitoring disease activity/occurrence ofcomorbidities in schizophrenia and/or multiple sclerosis by measuring25-hydroxyvitamin D3 level in a biological sample from a subject incombination with the levels of either or both IL-8 and MMP-9 in thebiological sample.
 10. A method for determining the efficacy of atreatment regimen for treating an inflammatory response, preferably inschizophrenia or multiple sclerosis in a subject by comparing the levelsof 25-hydroxyvitamin D3 in a biological sample from the subject,preferably in combination with the levels of least one biomarker whereinthe at least one biomarker is selected from IL-8 and MMP-9 in thebiological sample before, during and after treatment, wherein saidtreatment is considered efficient if the level of 25-hydroxyvitamin D3and/or IL-8 and/or MMP-9 is approaching a predetermined control levelfor these metabolites.
 11. A method according to claim 10 wherein thelevels of both IL-8 and MMP-9 in the biological sample are comparedbefore, during and after treatment.
 12. A method according to claim 10wherein determination of an efficient treatment is recognised bymeasuring a level of 25-hydroxyvitamin D3 that is above 50 nmol/L,preferably above 75 nmol/L relative to an initial measurement prior totreatment outside this range, and/or the level of IL-8 is preferablybelow 32 pg/ml and the level of MMP-9 is preferably below 705 ng/ml,preferably in the range 169705 ng/ml, relative to an initial measurementprior to treatment that is outside this range.
 13. A method forestablishing a reference biomarker profile comprising the steps of (a)determining a quantity of vitamin D3, preferably 25-hydroxyvitamin D3and at least one biomarker selected from IL-8 and MMP-9 in a sampleobtained from a healthy subject or a subject having an inflammatoryresponse, especially having schizophrenia or multiple sclerosis; and (b)storing the quantity of the vitamin D3, preferably 25-hydroxyvitamin D3and the selected biomarker in a reference biomarker profile respectivelyfor healthy subjects or subjects having an inflammatory response,especially having schizophrenia or multiple sclerosis.
 14. A kitcomprising means for detecting the level of 25-hydroxyvitamin D3 in abiological sample from a subject, preferably in combination with meansfor detecting the level of at least one biomarker wherein the at leastone biomarker is selected from IL-8 and MMP-9.
 15. A kit as claimed inclaim 14 wherein the kit comprises appropriate detection reagents andoptionally further additives.